Process for inhibiting the transfer of antibiotic resistance determinants in bacteria

ABSTRACT

PROCESS FOR INHIBITING THE TRANSFER OF ANTIBIOTIC RESISTANCE DETERMINANTS FROM DONOR BACTERIA TO RECIPIENT BACTERIA BY THE USE OF AN EFFETIVE AMOUNT OF A LINCOMYCIN ANTIBIOTIC.

United States Patent PROCESS FOR INHIBITING THE TRANSFER OF ANTIBIOTICRESISTANCE DETERMINANTS IN BACTERIA I Joanne Roeser, Kalamazoo, Mich.,assignor to The Upjohn Company, Kalamazoo, Mich.

No Drawing. Continuation-impart of application Ser. No. 749,584, Aug. 2,1968. This application Oct. 17, 1968, Ser. No. 768,502

Int. Cl. A61k 21/00 U.S. Cl. 424-481 7 Claims ABSTRACT OF THE DISCLOSUREProcess for inhibiting the transfer of antibiotic resistancedeterminants from donor bacteria to recipient bacteria by the use of aneffective amount of a lincomycin antibiotic.

CROSS REFERENCE TO RELATED APPLICATION This application is acontinuation-in-part of copending application Ser. No. 749,584, filed onAug. 2, 1968, and now abandoned.

BRIEF SUMMARY OF THE INVENTION The subject process concerns the use of alincomycin antibiotic, as herein defined, to prevent or inhibit thetransfer by conjugation of antibiotic resistance determinants from donorbacteria to recipient bacteria. Antibiotic resistance determinants aregenetic factors possessed by some bacteria which make them resistant tocertain antibiotics and chemical antibacterial agents, such as sulfadrugs. It is well known in the microbiological art that antibioticresistance determinants can be transferred from donor bacteria torecipient bacteria by conjugation. This transfer of resistancedeterminants is believed to be mediated through the agency of aresistance-transfer factor, abbreviated RTF. For example, if bacterium Apossesses a resistance determinant for tetracycline, and RTF is present,then by conjugation with bacterium B which does not possess such aresistance determinant, the latter bacterium may also become resistantto tetracycline. A recent comprehensive review of transferable drugresistance is published in Science Journal 4, pp. 71-76 (1968). Thecombination of resistance determinants and RTF is generally referred toas R-factor.

The process of the present invention effectively blocks the transfer ofresistance determinants from donor bacteria to recipient bacteria. Thus,for example, if an effective amount of a lincomycin antibiotic, defined,is used to treat the donor and recipient bacteria, described above, thenthe ability of the resistance determinant, possessed by bacterium A, tobe transferred to bacterium B is inhibited. The term donor bacteria ordonor means a bacterium which possesses a resistance determinant and'RTF; whereas the term recipient bacteria or recipient means a bacteriumwhich is not a donor but is capable of receiving a resistancedeterminant from a donor. The transfer of resistance determinants byconjugation has been shown to occur only between Gram-negative bacteria.

It should be noted that resistance determinants possessed by a bacteriumare not necessarily directed to a single antibiotic but often times asingle resistance determinant will enable the bacterium to be resistantto more than one antibiotic. Also, it is known that more than oneresistance determinant can be transferred during a single conjugation bythe presence of agency of a single RTF. Though the mechanism of thesubject process to inhibit the transfer of resistance determinants fromdonor to recipient bacterium is not completely understood, it is feltthat the 3,574,830 Patented Apr. 13, 1971 trated by the followingstructural formula:

CH3 R2 OAC1 wherein R is methyl, or ethyl; R is cis or trans alkyl of 2to 8 carbon atoms, inclusive; R is hydrogen or alkyl to 8 carbon atoms,inclusive; X is hydroxy, chlorine, bromine, or iodine, each in the (R)or (S) configuration; A0 and AC2 are hydrogen or acyl, selectedindependently, wherein acyl is the acyl of a hydrocarbon carboxylic acidof not more than 18 carbon atoms, or a halo-, nitro-, hydroxy-, amino-,cyano-, thiocyano-, or loweralkoxy hydrocarbon carboxylic acid of atotal of up to 18 carbon atoms; and, Ac is of the same group as Ac andAc or phosphate, and acid addition salts thereof.

The lincomycin compounds, herein defined, can be prepared by proceduresdisclosed in various patents, publications and patent applications.These are as follows:

LincomycinU.S. Pat. 3,086,912 with reference to Formula I, whereinR=methyl or ethyl-U.S. Pat. 3,380,992, (specification and Example 12) |R=cis or trans alkyl to 8 carbon atoms-US. Pat.

3,380,992, (specification and Example 1) R =Hydrogen or alkyl to 8carbon atomsU.S. Pat. 3,380,992, (specification and Examples 1E and 1Gand H) X: (S)OH-U.S. Pat. 3,380,992, (specification and Example 11 D)X=(R) or (S) C1 or BrBe1gium Pat. 676,202, US. Application Ser. No.498,989, filed Oct. 20, 1965, now abandoned X=(R) or (S) I-ApplicationSer. No. 696,518, filed Jan. 9, 1968, now US. Pat. 3,496,163 Lincomycinacylates-U.S. Pat. 3,326,981 Lincomycin 2-acylatesBelgium Pat. 696,412,US. Pat. application Ser. No. 568,102, filed July 27, 1966, now US. Pat.3,426,012 7-chlorolincomycin 2-acylates-U.S. pat. application Ser.

No. 637,358, filed May 10, 1967, now abandoned 7-chlorolincomycin2-phosphates and lincomycin 2-phosphates-US. Pat. application Ser. No.602,116, filed Dec. 16, .1966, now U.S. 'Pat. 3,487,068

Of the above compounds, the compound 7(S)-chloro- 7-deoxy-lincomycin isalso known by the generic name climmycin.

DETAILS OF THE INVENTION ple, upon contacting mating strains ofEscherichia coli, one strain of which harbors a resistance determinantfor tetracycline, with7(S)-chloro-7-deoxy-4depropyl-4'-pently-l'-demethyllincomycin in varyingconcentrations ranging from 0.5 ,ug./ml. to ,ug./ml., there is obtainedan Escherichia coli culture exhibiting a decrease in the frequency oftransfer of the tetracycline resistance determinant. Control runs, i.e.,wherein no lincomycin antibiotic is used, result in a high percentage ofconversion of the recipient bacteria from tetracycline sensitive totetracycline resistant due to R-factor transfer from the donorEscherichia coli bacteria.

A bacterial culture harboring Gram-negative bacteria, among which aredonors and recipients, can be contacted with a lincomycin antibioticpresent in various vehicles depending on the site of the bacterialculture. For example, for inhibiting the transfer of antibioticresistance determinants in bacteria resident in an animals gut, alincomycin antibiotic can be administered as an additive to a feed.Advantageously, the lincomycin antibiotic can be added to the feed as aliquid or solid premix composition in which the concentration of thelincomycin antibiotic is from about 100 to about 2000 times greater thanthe concentration of antibiotic desired in the final feed ration. Thelincomycin antibiotic can be dissolved or suspended in a fluid vehiclesuch as corn oil, cottonseed oil, water-based vehicles, molasses,distillers solubles, and the like, or in volatile solvents such asethanol, acetone, dimethylsulfoxide, dimethylformamide and the like, toprepare a liquid premix. Alternatively, a solid premix can be preparedby mixing the lincomycin antibiotic with an edible solid diluent such assucrose, lactose, starch, corn meal, flour, calcium carbonate, oystershell flour, soybean meal and the like.

It is common in the art of raising domestic animals to add growthstimulating agents, usually other antibiotic substances, for examplepenicillins, oxytetracycline, chlortetracycline, spectinomycin,bacitracin (various forms), erythromycin, tylosin, oleandomycin, and thelike, or non-antibiotic growth stimulating agents such as arsanilic acid(sodium salt), 3-nitro 4 hydroxyphenylarsonic acid, nitrofurazone,nitrofurazolidone, sulfamethazine and other sulfa drugs, trimethylalkylammonium stearate and the like to the feed or drinking water of theanimals. Such growth stimulating agents also increase the efficiency ofthe conversion of feed into animal weight. The lincomycin antibioticsthemselves possess growthpromoting qualities and can be added to thefeeds as the only antibiotic when growth stimulation is desired.Alternatively, a second growth-promoting agent can be fed concomitantlywith the lincomycin antibiotic. The second growth-promoting agent can beadded to the feed as a separate premix, for example as a commercialgrowthpromoting premix composition such as those readily available inthe marketplace. Advantageously, however, the lincomycin antibiotic anda second growth-promoting agent can be supplied in the same premixcomposition. Such premix compositions are then added to the animal feedto supply the lincomycin antibiotics at a rate of 10 to 1000 grams perton of feed. In the cases where the lincomycin antibiotics are to be fedfor their growthstimulating qualities in addition to theirRTF-suppressive qualities, levels at the higher end of the range aredesirable.

The lincornycin antibiotics, as herein defined, also can be administeredto the animals in their drinking water. When administered by this route,the lincomycin antibiotics in water-dispersible form can be added to thedrinking water as powders, crystals, premix solutions, premixsuspensions, tablets, effervescent tablets, syrups, or other convenientforms, at a rate to administer the lincomycin antibiotic in the range ofabout 10 to about 1000 mg. per gal. of drinking water.

For control of resistance determinant transfer, the subject lincomycinantibiotics can be administered systematically by injection, for exampleby injection into the blood or musculature of the animal, or orally. Inthe case of injection, the antibiotics can be administered in a rapidlydispersible form, for example, the lincomycin antibiotic can beadministered as readily soluble acidaddition salts, for example thehydrochloride or sulfate, or as readily soluble derivatives, for exampleas the ammonium 2-phosphate ester. In such administration, thelincomycin antibiotic is given in the range of about 1 to about mg. perkg. of body weight per day. Advantageously, in the case of intramuscularinjection, the lincomycin antibiotic can be given in a more slowlydispersible, long-acting depot form, for example as the pamoate(2,2-dihydroxy 1,1 dinaphthylmethane-3,3- dicarboxylic acid)acid-addition salt or as the Z-O-higher acylate ester, for example theZ-O-palmitate ester. For such depot injection a level of l to 100 mg.per kg. body weight is administered at infrequent intervals, for exampleonce or twice a week, to maintain a suitable blood level of thelincomycin antibiotic. For oral administration, the antibiotics may begiven in various oral dosage forms such as powders, pills, tablets,capsules, solutions, suspensions, oil-water emulsions and the like.

Lincomycin antibiotics, as herein defined, also can be administeredconcurrently to animals being given a second therapeutic agent intendedto combat an existing bacterial or viral infection. Such administrationof lincomycin antibiotics is advantageous in forestalling the transferof resistance determinants among surviving Gramnegative organisms. Forexample, the lincornycin antibiotic advantageously can be administeredconcurrently with agents such as penicillins, ampicillin, methicillin,nafcillin, oxacillin, phenecillin, tetracycline, chlortetracycline,oxytetracycline, demethylchlortetracycline, methacycline,rolitetracycline, doxycycline, erythromycin, oleandomycin, cephalothin,novobiocin, streptomycin, neomycin, kanamycin, cephaloglycin,cephaloridine, bacitra cin, polymyxin, spectinomycin, colistin,paromomycin, dihydrostreptomycin, dihydronovobiocin, sulfonamides,nitrofuran compounds, tylosin and moenomycin. The lincomycin antibioticand the second therapeutic agent can be administered concurrently as.separate medications, for example by separate injections or as separateoral medicaments, or they can be administered in suitable admixture witheach other, for example as a common single injection or in a single oralmedicament.

The word animals as used herein with reference to feeds includes suchanimals as cattle, swine, sheep, chickens, turkeys, other domestic fowl,dogs, cats, horses, and the like.

The compositions of the present invention are preferably presented foradministration to humans in unit dosage forms, such as tablets,capsules, pills, powders, granules, sterile parenteral solutions orsuspensions, and oral solutions or suspensions, and oil-water emulsionscontaining suitable quantities of a lincomycin antibiotic in the form ofthe free base, or pharmacologically acceptable salts and esters.

For oral administration, either solid or fluid unit dosage forms can beprepared. 'For preparing solid compositions such as tablets, theprincipal active ingredient is mixed with conventional ingredients suchas talc, magnesium stearate, dicalcium phosphate, calcium sulfate,starch, lactose, acacia, methylcellulose, and functionally similarmaterials as pharmaceutical diluents or carriers.

For parenteral administration, fluid unit dosage forms are preparedutilizing a lincomycin antibiotic and a sterile vehicle, Water beingpreferred. The compound, depending on the form and concentration used,can be either suspended or dissolved in the vehicle. In preparingsolutions, a water-soluble form of the lincomycin antibiotic can bedissolved in Water for injection and filter sterilized before fillinginto a suitable vial or ampul and sealing. Advantageously, adjuvantssuch as a local anesthetic, preservative and buffering agents can bedissolved in the vehicle.

To enhance the stability, the composition can be frozen after fillinginto the vial and the water removed under vacuum. The dry lyophilizedpowder is then sealed in the vial and an accompanying vial of water forinjection is supplied to reconstitute the solution prior to use.Parenteral suspensions are prepared in substantially the same mannerexcept that the compound is suspended in the vehicle instead of beingdissolved and sterilization cannot be accomplished by filtration. Thecompound can be sterilized by exposure to ethylene oxide beforesuspending in the sterile vehicle. Advantageously, a surfactant orwetting agent is included in the composition to facilitate uniformdistribution of the compound.

The dosage of a lincomycin antibiotic, as herein defined, depends onroute and frequency of administration; and the age, weight, andcondition of the patient. A dosage schedule of from about to 1000 mg, 1to 4 times daily (every six hours), embraces the effective range forinhibition of resistance determinant transfer. For children, the dosageis calculated on the basis of 5 to 30 mg./kg./ day to be administeredevery six hours.

The lincomycin antibiotic, as herein defined, is compounded with asuitable pharmaceutical carrier in unit dosage form for convenient andeffective administration. In the preferred embodiments of thisinvention, the dosage units contain the compounds in 15, 30, 50, 125,250, 350, 500, 750 andv 1000 mg. amounts for systemic use; in 0.25, 0.5,1.2 and 5% amounts for local use; and 5 to 65% w./v. for parenteral use.The dosage of compositions containing lincomycin antibiotic, as hereindefined, and one or more other active ingredients is to be determinedwith reference to the usual dosage of each such ingredient.

The following examples are illustrative of the process and products ofthe present invention but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 An Escherichia coli donor strain harboring a resistancetransfer factor with determinants for resistance to tetracycline (25ng./ml.), chloramphenicol (25 ug./ml.), streptomycin (25 ag./ml.), andsulfathiazole (100 ,ag./ml.) is inoculated by a loop from a PenAssayAgar slant into PenAssay Broth with or without an appropriateconcentration of one of the following lincomycins: 7(8)- chloro 7 deoxy1'-demethyllincomycin as the hydrochloride salt (U26,285A); 7(S) chloro7 deoxylincomycin as the hydrochloride hydrate salt (U-21,251F-); and 7(S) chloro 7-deoxy-4'-depropyl-4'-pentyl-l'-demethyllincomycin as thehydrochloride salt (U-24,729A). U26,285A and U-21,251F are tested atconcentrations ranging from 0.5 ag/ml. to 200 g./ml.; whereas U-24,729Ais tested at concentrations ranging from 0.5 ,ug/ml. to ag/ml. The donorbroth cultures are incubated at 37 C. for 24 hours on a reciprocatingshaker.

An Escherichia coli recipient strain, which harbors no resistancetransfer factor, and is sensitive to tetracycline (25 ag./ml.), andcarries a chromosomal resistance to streptomycin (750 g./ml.), isinoculated by a loop from a PenAssay Agar slant into PenAssay Broth andis incubated at 37 C. for 21 hours on a reciprocating shaker. Therecipient culture is diluted in PenAssay Broth and is incubated at 37 C.to achieve a logarithmically growing culture at a concentration of 2 to5 10 cells per ml.

Mating mixtures are prepared by adding 0.5 ml. of donor culture to 5.0ml. of recipient culture to which an appropriate concentration of thelincomycin antibiotic, as given previously, was added in order tomaintain the same concentration in the mating mixture as in eachpre-treated donor culture respectively. The mating mixtures areincubated at 37 C. for 1 hour with gentle shaking.

Viable counts of the donor and recipient cultures are made at 0 time ofthe mating experiment by diluting samples in Spizizens minimal saltssolution and by plating aliquots of each solution on PenAssay Agarplates. The numbers of converted recipients [recipients resistant totetracycline (25 g./ml.)] are determined by making dilu tions of eachmating mixture in Spizizens minimal salts solution and by platingaliquots of each dilution on Pen- Assay Agar plates containing bothtetracycline (25 ,ug./ ml.) and streptomycin (750 ,ag./ml.). The platesare incubated at 37 C. for 24 hours before the number of colonies isascertained.

Calculations are made to determine the number of converted recipientsper input donor cell. A negative number is obtained even for controlcultures since few cells in the total donor population are capable oftransfer at any given time.

The data presented in the following table give the number of convertedrecipients per input donor cell at each concentration of the lincomycincompound tested. These same data when graphed show that an exponentialdecrease in the frequency of transfer of the tetracycline resistancedeteminant occurs with a linear increase in the concentration of each ofthe lincomycins tested.

The PenAssay Agar disclosed above is prepared by using PenAssay Broth(Difco Antibiotic Medium No. 3, Difco, Detroit, Mich.) plus 15grams/liter of agar (Difco).

PenAssay Broth consists of Difco antibiotic Medium No. 3.

Spizizens minimal salts solution contains the following in g./liter ofdeionized water:

K HPO 14.0; KH PO 6.0; sodium citrate, 5H O, 1.0;

and (NH4)2SO4, 2.0.

TABLE I.-EFFEC[ OF LINCOMYOIN ANTIBIOTICS ON RESISTANCE TRANSFER Numberof converted recipients per initial donor roll U24,720A U-21,251FU26,285A

2. 1X10" 1. 3X10- 1. 2 (l.0 8. 3X10- 1. 9X10- 4. 9X10- 2. 2X10- 8. 9X10-*The degree to which low frequencies of transfer can be measured isdependent both on the initial donor viable count and the number ofconverted recipients which can be detected. Thus the limitation of themeasurement varies from one culture to another.

EXAMPLE 2 Clinimycin, (7(S)-chloro 7 deoxylincomycin) (U- 21,251F) and7(.S)-chloro 7 deoxy-4-depropyl-4- pentyl-1-dimethyllincomycin,(U-24,729A) have been shown to inhibit resistance transfer in vivo inthe chick using the following experimental system:

Escherichia coli CSH-2 which harbors a resistance transfer factor withdeterminants for resistance to tetracycline 25 .ag./ml.),chloramphenicol 25 g./ml.), streptomycin (25]ag./ml.), and sulfathiazolemg./ ml.) is employed as the donor strain. An Escherichia coli strainwhich harbors no resistance transfer factor, is sensitive to tetracyline25 ,ug./Inl.), and carries a chromosomal resistance to streptomycin (750,ag/ml.) is used as the recipient strain. The donor and recipientstrains are grown overnight at 37 C. in PenAssay Broth (Difco). Therecipient culture is concentrated six-fold. Both the bacterial culturesand the compounds, clinimycin and 7(S)-chloro-7- deoxy-4-depr0pyl 4pentyl-l'-demetl1yl1incomycin dissolved in deionized water, areadministered to the chicks in 0.5 ml. doses by oral intubation. Groupsof chicks, each chick weighing approximately 0.035 kg., are treated withvarious concentrations of the compounds.

On Day 1, oneday old chicks are given the donor strain of E. coliorally. This is followed hours later by the first oral dose ofclinimycin or 7(S)-chloro-7-deoxy-4'-depropyl-4'-pentyl-1-demethyllincomycin. On Day 2, two doses of eitherclinimycin or 7(,S)-chloro 7 deoxy-4'- depropyl-4'-pentyl 1'demethyllincomycin are administered approximately 7 hours apart. Midwaybetween these two doses, the chicks are given the concentratedsuspension of the recipient strain. The chicks are sacrificed on Day 3.Both donor and recipient E. coli are present simultaneously in the gutfor approximately 20 hours.

The large intestine is removed from each chick into a sterile Petridish. The gut is perforated with a dissecting knife and washed with 2ml. of PenAssay Broth. The resulting suspensions are diluted and platedon PenAssay Agar with tetracycline (25 ,ug/ml.) to determine the numberof donor cells; on PenAssay Agar with strep-tomycin (750 ,ug./ ml.) todetermine the number of recipient cells; and on PenAssay Agar withtetracycline (25 g/ ml.) and streptomycin (750 ig/ml.) to determine thenumber of converted recipients (recipients resistant to tetracycline) ineach gut sample. The plates are incubated at 37 C. for 24 hours beforecolony counts are made.

The data are given in the following Table II for clinimycin and in TableIII for 7(S)-chloro-7-deoxy-4-de propyl-4'-pentyl-1-demethyllincomycin.The degree of inhibition of resistance transfer is dependent on theconcentration of each compound used. A concentration of 100 mg./ kg. ofclinimycin completely suppresses resistance transfer in the chicks asdoes a 50 mg./kg. dosage level of 7(S)-chloro-7-deoxy 4depropyl-4'-pentyl-l'-demethyllincomycin.

*Base equivalent of clinimycin.

TABLE lII.7(S)-OHLORO-DEOXY-47-DEPROPYL-4.-PEN- TYIrY-DEMETHYLLINCOMYCIN(II-24,729A) Converted Donors Recipients recipients Mean IA 2. 9x10 7.7x10 4. 3x10 IB 3. 5x10 3. 7X10 1. 3x10 1 Ding/kg IC 6.8)(10 3. 0X101.0)(10 1. 6x10 ID 2.3)(10 2.8Xl0 3.8)(10 J IE 1. 6X10 5. 6X10 2. 5X10HA 1. 1X10 2. 8X10 1. 1X10 HR 3. 6X10 1. 1X10 2X10 3.125 mg./kg HO 4.6X10 1. 4X10 4. 5X10 2. 4x10 IID 6. 4X10 3.1)(10 7. 0X10 HE 4. 1X10 3.9x10 1. 2x10 IIIA 1.2)(10 5. 2x10 5 1X10 IIIB 1. 2x10 6. 2X10 1. 4X106.25 mg./kg H10 7. 6x10 4. 1X10 2. 6X10 9. 6X10 IIID 2. 4X10 2. 8X10 94x10 1 IIIE 6. 4X10 24x10 1 7X10 IVA 8. 4x10 2. 6X10 2. 5X10 IVB 7. 5x102. 2X10 1. 2X10 12.5 lug/kg"--. WC 9. 7X10 2. 2X10 1. 0X10 5. 5X10 IVD5. 6x10 5. 2x10 2. 0 10 J IVE 8. 3x10 1. 9X10 8. 5X10 VA 3. 9X10 5.4X10 1. 0X10 VB 3. 5x10 8. 1x10 1. 0X10 25 rug/kg VC 4.2)(10 1.6)(10 7.5X10 1. 0X10 VD 2. 7X10 2. 3X10 1. 0X10 VE 1. 9x10 5. 5X10 4. 1X10 [A 2.8X10 1. 9X10 1. OXlO VIB 4. 3X10 8. 0x10 1. 0X10 50 mg./kg VIC 4. 0X102. 4X10 1. 0X10 1. 0X10 VID 4.1)(10 7. 1X10 1. 0X10 VIE 1. 5X10 1. 9 l01.0X10

EXAMPLE 3 Sterile powder A completely soluble sterile powder ofclinimycin hydrochloride which, on reconstitution, can be administeredeither parenterally (I.M., I.V. or subcutaneously) or topically is putup in sterile vials, each vial containing 1 gm. of antibiotic. For use,water for injection or sterile normal saline is added to give a finalvolume of 10 ml. This results in a 10% W./v. concentration or mg. ofantibiotic per m1. of reconstituted product. The preparation ofclinimycin hydrochloride is disclosed in Belgium Pat. 676,154.

EXAMPLE 4 Intramuscular depot-form injectable for animals A sterileaqueous suspension suitable for intramuscular injection and containingin each ml. 400 mg. of clinimycm pamoate is prepared from the followingingredients:

Gm. Clinimycin pamoate, micronized 400 Polyvinylpyrrolidone 5 Sodiumcarboxymethylcellulose, low visc. 1 Sodium citrate, hydrous 8Methylparaben 1.5 Propylparaben 0.1

Water for injection q.s. 1000 ml.

The preparation of clinimycin pamoate is disclosed in Belgium Pat.676,154.

EXAMPLE 5 T ablets for large animals 10,000 tablets for oraladministration to large animals such as cattle or horses and containingin each tablet 1000 mg. of clinimycin hydrochloride are prepared fromthe following ingredients:

The ingredients are mixed thoroughly and slugged. The ingredients arebroken down by passing through a number sixteen screen. The resultinggranules are then compressed into tablets each containing 1000 mg. ofclinimycin hydrochloride.

EXAMPLE 6 Premix compositions PREMIX A Clinimycin hydrochloride20 gm.Lactose-qs. 1 lb.

PREMIX B 1-demethylclinimycin hydrochloride 60 gm. Corn mealq.s. 1 lb.

1 Preparation disclosed in Belgian Pat. 676,154.

PREMIX C 4'-pentyl-1'-demethyl analog of clinimycin hydrochloride gm.Wheat flourq.s. 1 lb.

PREMIX D Clinimycin base- 20 gm. Cottonseed oil-1 lb.

PREMIX E 4'-pentyl-1-demethyl analog of clinimycin hydrochloride20 gm.Waterl liter PREMIX F Lincomycin-Z-acetate --50 gm. Ethyl acetate-1liter Disclosed in U.S. Pat. 3,326,891, Example 6.

PREMIX G Clinimycin pamoate20 gm. Bacitracin zinc-25 gm. Cornq.s. 1 lb.

PREMIX H 4'-penty1-1'-demethyl analog of clinimycin hydrochloride-10 gm.Oxytetracycline quaternary salt-50 gm. Sucrose-q.s. 1 lb.

PREMIX I l-demethylclinimycin hydrochloride50 gm. Chlortetracyclinehydrochloride50 gm. Oyster shell flourq.s. 1 lb.

PREMIX J 4' pentyl 1' demethyl analog of clini'mycin, 2-

palmitate ester --10 gm.

Procaine penicillin G20 gm.

Preparation disclosed in U.S. patent application Ser. No. 637,358, filedMay 10, 1967.

The foregoing premix compositions are prepared from finely ground solidmaterials by mixing all ingredients 10 together in an appropriatemanner. In the case of liquid premix compositions the solids aredissolved or suspended in the liquids by methods known in the art.

Following the preceding formulas, premixes are simi larly preparedsubstituting other compounds of formula 1 for those given. In thepremixes where a second antibiotic is present, as for growth promotingor therapeutic purposes, the second antibiotic may be taken from thoseknown for growth promoting or therapeutic purposes in the art, forexample those listed in the specifications above.

The above premix compositions are added to the feed of the animal, or(premix E) to the drinking water, in an amount to supply the antibioticsat the rate specified above.

EXAMPLE 7 Swine growing diet for hogs of 40 to pounds body weight 0.08;Fe, 5.0 Cu, 0.4;1, 0.24 Zn, 0.7.

2 Contains 800 USP units of Da/gm. and 1500 LU. of A/gm.

Contains per 1b.: Riboflavin, 200 mg.; calcium. pantothenate, 4000 mg.;niacin, 9000 mg.; and choline chloride, 10,000 mg.

4 Contains 6 mg. B12 per lb.

To 99 parts of the preceding feed is added 1 part of Premix A to providea feed with 200 mg. of the lincomycin antibiotic per lb. of feed.

Substituting Premixes B through I, feeds are prepared with varyingamounts of lincomycin antibiotics, with or without a secondgrowth-stimulating or therapeutic antibiotic. The foregoing compositionis usefully fed to hogs for increased weight gain and improved feedconversion efficiency, and inhibition of transfer of resistancedeterminants.

EXAMPLE 8 A fattening feed for 800 pound yearling cattle is preparedfrom the following types and amounts of ingredients:

Ground ear corn 89.75 Soybean oil meal, 44% 9.0 Ground limestone 0.7Sodium chloride 0.5 Trace mineral mixture 1 0.05

Contains the following percent of minerals: Mn, 12; Co, 0.08 Fe, 5.0;Cu, 0.4 I, 0.24 Zn, 0.7.

To 99 parts of the preceding feed is added 1 part of Premix A to providea feed with 200 mg. per lb. of antibiotic.

Substituting Premixes B through I, inclusive, feeds are prepared withvarying amounts and ratios of antibiotics.

Cattle are to receive the foregoing feed ad libitum together with 5 lb.of hay per head per day for an increased rate of weight gain, increasedfeed efficiency, and inhibition of transfer of resistance determinants.

1 1 EXAMPLE 9 A chicken feed for broilers is prepared from the followingtypes and amounts of ingredients:

Percent Yellow corn meal 67.35 Soybean oil meal, 50% 24.00 Menhaden fishmeal, 60% 6.00 Steamed bone meal 1.00 Ground limestone 1.00 Iodized salt.34 25% Choline chloride .13 Vitamin B supplement (6 mg./ lb.) .10Manganese sulfate .02 Supplemental vitamin mix 1 .06

Consisting of 16.0 gm. vitamin A supplement (10,000 units/gm.); 3.6 gm.vitamin D3 supplement (15,000 units/ gm.); 7.1 gm. riboflavin supplement(1 gm. riboflavin per ounce) 500 mg. niacin.

To 99 parts of the preceding feed is added 1 part of Premix A to providea feed with 200 mg. per lb. of antibiotic.

Substituting Premixes B through I, inclusive, feeds are prepared withvarying amounts and ratios of antibiotics.

The foregoing composition is usefully fed to chickens for increased rateof weight gain, improved utilization of feed, and inhibition of transferof resistance determinants.

EXAMPLE 10 Drinking water for chickens, turkeys, ducks, geese or otherdomestic fowl Premix E is added to the drinking water of the animals toprovide 10 to 1000 mg. of trans-4-pentyl-l-demethyl analog of clinimycinhydrochloride antibiotic per gal.

Other lincomycin antibiotics of Formula 1 may be substituted for thatgiven in Premix E, to provide protection against transfer of resistancedeterminants.

EXAMPLE 1 1 Mastitis ointment One thousand grams of an ointment for theinhibition of transfer of resistance determinants in dairy cattle beingtreated for mastitis is prepared from the following types and amounts ofingredients:

Gm. Clinimycin hydrochloride 25 Methylprednisolone acetate 0.5 Lightliquid petrolatum 300 Chlorobutanol, anhydrous 5 Polysorbate 80 5 2%aluminum monostearate-peanut oil gel 400 White petrolatum q.s. 1000 gm.

The clinimycin hydrochloride and methylprednisolone acetate are milledwith the light liquid petrolatum until finely divided and uniformlydispersed. The chlorobutanol, polysorbate 80, peanut oil gel and whitepetrolatum are heated to 120 F. to form a melt and the liquid petrolatumdispersion stirred in. With continued stirring, the dispersion isallowed to cool (and congeal) to room temperature and is filled intodisposable mastitis syringes in gm. doses.

The above formulation can be administered to cattle exposed to cattleafiiicted with mastitis in order to prevent the non-afllicted cattlefrom receiving Gram-negative bacteria possessing resistance determinantsto various antibiotics and chemical antibacterial agents, such as sulfadrugs.

EXAMPLE 12 Capsules One thousant two-piece hard gelatin capsules fororal use, each containing 250 mg. of clinimycin hydrochloride 12 areprepared from the following types and amounts of materials:

Gm. Clinimycin hydrochloride 250 Corn starch Talc 75 Magnesium stearate25 The materials are thoroughly mixed and then encapsulated in the usualmanner.

The foregoing capsules are useful for the inhibition of transfer ofresistance determinants in adult humans by the oral administration of 1capsule every 4 hours.

Using the procedure above, capsules are similarly prepared containingclinimycin hydrochloride in 25, 50, 125, 350 and 500 mg. amounts bysubstituting 25, 50, 125, 350 and 500 gm. of clinimycin hydrochloridefor the 250 gm.

used above.

EXAMPLE 13 Capsules One thousand two-piece hard gelatin capsules fororal use, each containing 50 mg. of the 4-pentyl-l'-demethyl analog ofclinimycin hydrochloride and 250 mg. of tetracycline hydrochloride, areprepared from the following types and amounts of ingredients:

4'-pentyl-1'-demethyl analog of clinimycin hydrochloride 50 Tetracyclinehydrochloride 250 Talc 75 Magnesium stearate 25 The ingredients arethoroughly mixed and then encapsulated in the usual manner.

The foregoing capsules are useful for the inhibition of transfer ofresistance determinants in adult humans by the oral administration of 1capsule every 6 hours.

Using the procedure above, capsules are similarly prepared containingthe 4'-penty1-1'-demethyl analog of clinimycin hydrochloride and each ofthe following antibiotics in place of tetracycline by substituting 250gm. of such other antibiotic for tetracycline: penicillins, ampicillin,methicillin, nafcillin, oxacillin, phenecillin, chlortetracycline,oxytetracycline, demethylchlortetracycline, metacycline,rolitetracycline, doxycycline, erythromycin, oleandomycin, cephalothin,kanamycin, novobiocin, streptomycin, neomycin, cephaloglycin,cephaloridine, bacitracin, polymyxin, spectinomycin, colistin,paromomycin, dihydrostreptomycin, dihydronovobiocin, sulfonamides,nitrofuran compounds, chloramphenicol, fumagillin, tylosin andmoenomycin. When a penicillin, such as potassium penicillin G, is to beused in place of tetracycline, 250,000 units per capsule is employed.

Such combination products are useful for the inhibition of transfer ofresistance determinants in mixed infections in adult humans by the oraladministration of 1 capsule every 6 hours.

EXAMPLE 14 Tablets One thousand tablets for oral use, each containing500 mg. of clinimycin hydrochloride are prepared from the followingtypes and amounts of materials:

Gm. Clinimycin hydrochloride 500 Lactose Corn starch 65 Magnesiumstearate 25 Light liquid petrolatum 3 The ingredients are thoroughlymixed and slugged. The slugs are broken down by forcing through a numbersixteen screen. The resulting granules are then compressed into tablets,each tablet containing 500 mg. of clinimycin hydrochloride.

13. The foregoing tablets are useful for the inhibition of transfer ofresistance determinants in infections in adult humans by oraladministration of 1 tablet every 4 hours.

Using the above procedure, except for reducing the' amount of clinimycinhydrochloride to 250 gm., tablets containing 250 mg. of clinimycinhydrochloride are prepared.

EXAMPLE 15 Tablets One thousand oral tablets, each containing 250 mg. ofclinimycin hydrochloride and a total of 250 mg. (83.3 mg. each) ofsulfadiazine, Sulfamerazine, and sulfamethazine, are prepared from thefollowing types and amounts of materials:

The ingredients are thoroughly mixed and slugged. The slugs are brokendown by forcing through a number sixteen screen. The resulting granulesare then compressed into tablets, each containing 250 mg. of clinimycinhydrochloride and a total of 250 mg. (83.3 mg. each) of sulfadiazine,Sulfamerazine, and sulfamethazine.

The foregoing tablets are useful for the inhibition of transfer ofresistance determinants in infections by the oral administration of 4tablets first and then 1 every six hours.

For the inhibition of transfer of resistance determinants in urinaryinfections, the triple sulfas in the above formulation is advantageouslyreplaced by 250 gm. of sulfamethylthiadiazole or 250 gm. ofsulfacetamide.

EXAMPLE 16 Oral syrup One thousand ml. of an aqueous suspension for oraluse, containing in each 5-ml. dose, one-half gram of total sulfas and250 mg. of clinimycin hydrochloride is prepared from the following typesand amounts of ingredients:

Clinimycin hydrochloride gm 50 Sulfadiazine gm 33.3 Sulfamerazine gm33.3 Sulfamethazine gm 33.3 Citric acid gm 2 Benzoic acid gm 1 Sucrosegm 700 Tragacanth gm 5 Lemon oil ml 2 Deionized water, q.s. 1000 ml.

The citric acid, benzoic acid, sucrose, tragacanth, and lemon oil aredispersed in suflicient Water to make 850 ml. of solution. Theclinimycin hydrochloride and finely powdered sulfas are stirred into thesyrup until uniformly distributed. Suflicient Water is added to make1000' ml.

The composition so prepared is useful for the inhibition of transfer ofresistance determinants at a dose of 1 teaspoonful (5 ml.) 4 times a dayin adult humans being treated systemically for pneumonia.

EXAMPLE 1? By substituting 150 gm. of lincomycin hydrochloride forclinimycin hydrochloride in Example 16, an oral syrup containingone-half gram of total sulfas and 750 mg. of lincomycin hydrochloride ineach S-ml. dose is prepared.

The composition so prepared is useful for the inhibition of transfer ofresistance determinantsat a dose of 1 tea- 14 spoonful (5 ml.) 4 times aday in adult humans being treated systemically for pneumonia.

EXAMPLE l8 Parenteral solution A sterile aqueous solution forintramuscular use, containing in 1 ml. 200 mg. of clinimycinhydrochloride is prepared from the following types and amounts ofmaterial-s:

Gm. Clinimycin hydrochloride 200 Lidocaine hydrochloride 4 Methylparaben2.5 Propylparaben 0.17

Water for injection, q.s. 1000 ml.

The ingredients are dissolved in the Water and the solution sterilizedby filtration. The sterile solution is filled into vials and the vialssealed.

EXAMPLE 19 Parenteral preparation A sterile aqueous solution forintramuscular use, containing in 1 ml. 200 mg. of clinimycinhydrochloride and 400 mg. of spectinomycin sulfate, is prepared from thefollowing types and amounts of ingredients:

Gm. Clinimycin hydrochloride 2 00 Spectinomycin sulfate 400 Lactose 50Water for injection, q.s. 1000 ml.

EXAMPLE 20 Cream One thousand grams of a vaginal cream are prepared fromthe following types and amounts of ingredients:

Gm. Clinimycin hydrochloride 50 Tegacid Regular 150 Spermaceti Propyleneglycol 50 Polysorbate 8O 5 Methylparaben 1 Deionized water, q.s. 1000gm.

1 Self-emulsifying glyceryl monostearate from Goldschmidt ChemicalCorporation, New York, N.Y.

The Tegacid and spermaceti are melted together at a temperature of70-80" C. The methylparaben is dissolved in about 500 gm. of Water andthe propylene glycol, Polysorbate 80, and clinimycin hydrochloride areadded in turn, maintaining a temperature of 7580 C. The methylparabenmixture is added slowly to the Tegacid and Spermaceti melt, withconstant stirring. The addition is continued for at least 30 minuteswith continued stirring until the temperature has dropped to 40- 45 C.The pH of the final cream is adjusted to 3.5 by incorporating 2.5 gm. ofcitric acid and 0.2 gm. of dibasic sodium phosphate dissolved in about50 gm. of water. Finally, sufficient Water is added to bring the finalweight to 1000 gm. and the preparation stirred to maintain homogeneityuntil cooled and congealed.

The foregoing composition is useful for the inhibition of transfer ofresistance determinants in conjunction with the treatment of vaginalinfections in humans.

EXAMPLE 21 Ointment, ophthalmic One thousand grams of an ophthalmicointment containing 0.5% clinimycin hydrochloride are prepared from thefollowing types and amounts of ingredients:

Gm. Clinimycin hydrochloride 5 Bacitracin 12.2 Polymyxin B sulfate(10,000 units/mg.) 1 Light liquid petrolatum 250 W001 fat 200 Whitepetrolatum, q.s. 1000 gm.

The solid ingredients are finely divided by means of an air micronizerand added to the light liquid petrolatum. The mixture is passed througha colloid mill to uniformly distribute the micronized particles. Thewool fat and white petrolatum are melted together, strained, and thetemperature adjusted to 45-50 C. The liquid petrolatum slurry is addedand the ointment stirred until congealed. Suitably the ointment ispackaged in one dram ophthalmic tubes.

The foregoing ointment is usefully applied to the eye for the inhibitionof transfer of resistance determinants in humans and other animals.

Advantageously the foregoing composition can contain 5 gm. (0.5%) ofmethylprednisolone for the treatment of inflammation, and,alternatively, the bacitracin and polymyxin B sulfate can be omitted.

EXAMPLE 22 Eye-ear drops One thousand cc. of a sterile aqueous solutionfor eye or ear use containing mg. of clinimycin hydrochloride and 5 mg.of methylprednisolone phosphate sodium in each ml. is prepared from thefollowing types and amounts of ingredients:

Clinimycin hydrochloride 10 Methylprednisolone phosphate sodium 5 Sodiumcitrate 4.5 Sodium bisulfite l Polyethylene glycol 4000 120 0.2

Myristyl-y-picolinium chloride Polyvinylpyrrolidone 1 Deionized water,q.s. 1000 ml.

bacteria to mating Gram-negative recipient bacteria which comprisescontacting said donor and recipient bacteria with an effective amount ofa lincomycin antibiotic of the formula:

R2 IL CH3 a H 51 (Ham. 1 1', AczO o OAB1 OAcs wherein R is methyl orethyl; R is cis or trans alkyl of 2 to 8 carbon atoms, inclusive; R ishydrogen or alkyl of 1 to 8 carbon atoms, inclusive; X is hydroxy,chlorine, bromine, or iodine, each in the (R) or (S) configuration; Acand A0 are hydrogen or acyl, selected independently, wherein acyl is theacyl of a hydrocarbon carboxylic acid of not more than 18 carbon atoms,or a halo-, nitro-, hydroxy-, amino-, cyano-, thiocyan0-, or loweralkoxyhydrocarbon carboxylic acid of a total of up to 18 carbon atoms; and Acis of the same group as AC1 and Ac or phosphate, or acid-addition saltsthereof.

2. A process, according to claim 1, wherein the lincomycin antibiotic islincomycin free base or an acid addition salt thereof having thefollowing structure:

3. A process, according to claim 1, wherein the lincomycin antibiotic isa chlorinated lincomycin antibiotic.

4. A process, according to claim 1, wherein the lincomycin antibiotic is7(S)-chloro-7-deoxylincomycin.

5. A process, according to claim 1, wherein the lincomycin antibiotic is7(S)-chloro-7-deoxy-1-demethyllincomycin.

6. A process, according to claim 1, wherein the lincomycin antibiotic is7 (S)-chloro-7-deoxy-4'-depropy1- 4-pentyll '-demethyllincomycin.

7. A process, according to claim 1, wherein said efiective amount of alincomycin antibiotic is in the range of from about 0.5 to about ng./ml.

References Cited UNITED STATES PATENTS 3,086,912 4/1963 Bergy et a1.424-181 3,380,992 4/1968 Argoudelis et a1. 260-210 OTHER REFERENCESKawakami et al., Biochemical and Biophysical -Re search Communications,vol. 18, Nos. 5-6, 1.965, pp. 716724.

ALBERT T. MEYERS, Primary Examiner D. M. STEPHENS, Assistant ExaminerUNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,574,850 Dated Apr i l 13, 1971 Inventofls) Joanne Roese" It is certifiedthat error appears in the above-identified patem and that said LettersPatent are hereby corrected as shown below:

Col umn 1, i ine 52, for ant ib iotic, defined, read ant i biotic, ashere in defined i ine 69, for "of" read or Coi umn 5, i ine 28, for "1.2" read 1,2 Col umn 6, i ine 5, for "(25 9 ./mi read (25 gg/mi i ine22, for "deteminant' read determinant i ine +1, for "roi 1 read cel i iine 65, for "d imethyl i incomyc read demethyl i incomyc in i ines73-74, for tetracyi ine" read tetracyci ine Col umn 7, I ine 57, for i"1 .0 read 1 .0 Col umn 8, I ine 1, for "chloro-deox i'T' readchioro-T-deoxy-4' Coi umn 9, I ine 52, for "corn-q.s read corn meal q.sColumn 10,

i ine 37, For "200 mg. read 2000 mg Coi umn 11, i ine 7 L, for"thousant" read thousand Signed and sealed this 31st day of August 1971(SEAL) Attest:

EDWARD M.FLETCHER, JR. WILLIAM E. SCHUYLER, JR. Attesting OfficerCommissioner of Patents

